The tumor antigen (T-antigen) encoded by the viral A gene of SV40 is a large, molecular weight phosphoprotein that is expressed in cells lytically infected with and transformed by SV40. T-antigen is thought to be responsible for regulating both viral DNA replication and transcription as well as for initiating and maintaining the transformed pehnotype in a variety of cells. The aim of this research is to understand how T-antigen carries out these diverse and biologically significant functions. We recently developed an in vitro transcription system to test the affect of T-antigen on the expression of early and late SV40 genes. Our findings indicate that purified T-antigen binds specifically to SV40 DNA at three closely spaced sites and acts as a repressor to inhibit selectively the transcription of viral early RNA. We now propose to investigate the precise mechanism of this transcriptional regression. First, we will map by in vitro mutagenisis the region of SV40 DNA required to promote transcription of early and late genes both in vitro and in vivo. Next, we will attempt to purify RNA polymerase II and its selectivity factors in order to perform specific holoenzyme DNA binding studies at the promoter sequences. The mechanisms of transcriptional selectivity provided by the specificity determinants will be tested in vitro both by binding studies and by transription. Finally, we will study the interaction T-antigen with RNA polymerase and its specificity factors at the early and late promoter-binding sites. In addition to studying the regulation of transcription by T-antigen, we will also continue our studies with the involvement of T-antigen in viral DNA replication. Several recent studies have indicated that a direct interaction between T-antigen and the viral orgin of DNA replication may be a prerequisite to initiating DNA synthesis. We hope to develop an in vitro replication system that will be dependent on T-antigen for initiating viral replication at the SV40 origin. Such a system will provide a useful tool for investigating the mechanism by which T-antigen initiates viral DNA synthesis. We will also continue our studies of the interaction between the SV40 A gene product and specific DNA sequences and polypeptides of the host cell as a means of investigating the role of T-antigen in virally induced cellular transformation.